A detailed understanding of the molecular mechanism of protein synthesis is valuable both because of its importance in cellular metabolism and because of the possibility that gene expression may be controlled in part at the level of translation. We shall concentrate on the initiation phase of protein synthesis since translational controls are most likely to be found there. We propose to study in vitro the initiation process in rabbit reticulocytes and to elucidate its molecular mechanism at the enzymological level. The approach is first to isolate, identify and purify all of the macromolecular components involved in initiation, in particular to prepare homogeneous initiation factors. Second, we intend to study the chemistry and function of the components and to elucidate the pathway and enzymology of their interactions during initiation. We shall use crosslinking techniques to examine the subunit structure of the large initiation factor complex. IF-3, and to determine (in collaboration with Dr. R.R. Traut) which ribosomal proteins are involved in the binding of initiation factors to ribosomes. Third, we propose to construct from the purified components a fully defined cell-free system capable of efficient initiation of protein synthesis with natural mRNA. Its construction will allow us to examine at the molecular level exactly how rates of initiation are controlled, how specific classes of mRNA's may be selected for translation, or how possible covalent modification (for example phosphorylation) of initiation factors may play a role in the regulation of translation. Since most of the components of initiation appear to be interchangeable with those from different eucaryotic cell types, this system will enable us to study a great variety of control problems. The elucidation of the mechanism of initiation of protein synthesis will help provide a sound basis for further understanding of aberrations of function or control which may occur in diseased or malignant cells.